The goal of the proposed research is to delineate the functions in vivo of proteins that participate in splicing regulation. Using the example of sex-specific splicing regulation of the Drosophila binary switch gene Sex-lethal (Sxl), studies have shown that SANS-FILLE (SNF), the counterpart of the human U1 snRNP-U1A and U2 snRNP-U2B" proteins, plays a key role in Sxl splicing autoregulation. While it is clear that SNF and the female-specific SXL protein physically interact and are found in the same multi-component complex, the identity of the other members of this complex and how they are assembled to inhibit splicing of the male-specific exon remains to be determined. To address these issues, three specific aims are proposed. In Aim 1, the hypothesis, based on data from genetic and protein-protein interaction studies, that SPP-87B and SIN play integral roles in Sxl splicing autoregulation will be tested by a combined approach that will include protein localization studies, protein-protein interaction studies, and genetic analysis of loss-of-function mutations. In Aim 2, additional proteins that function in Sxl splicing autoregulation will be identified by exploiting information from studies in yeast and mammalian cells to create mutations in orthologous splicing factors and testing them for both genetic interactions and failure to consistently skip the Sxl male-exon. Candidate loci that have not yet been connected to splicing will be identified in a genome-wide genetic screen coupled with biochemical verification. In Aim 3, the biochemical properties of SNF and its partner proteins important for female-specific Sxl splicing will be identified using protein-protein and protein-RNA interaction assays. In addition, the molecular pathway that controls male-exon utilization will be elucidated by identifying which interactions are dependent on the presence of SXL and/or the association between SXL and SNF using extracts from mutant animals. The combined data from these three aims will provide new insight into how SXL, SNF and their protein partners are assembled into a blocking complex and function together to promote male-exon skipping. More importantly, because of the remarkable conservation of known splicing factors between Drosophila and humans, the information gained from these studies will significantly advance our understanding of regulated splicing in vertebrates.